Methods for production of arrays with modified oligonucleotide and polynucleotide compositions

ABSTRACT

The present invention provides methods for producing arrays having associated modified nucleic acid structures, e.g. acid stable and/or end-blocked nucleic acids such as 2′-O—R oligonucleotides. In one embodiment, arrays produced using the methods of the invention exhibit an increased binding affinity with complementary nucleic acids, and in particular with complementary RNA. In another embodiment, the associated nucleic acids of the array produced using the methods of the invention exhibit substantial acid resistance, allowing the arrays to be treated with low pH solutions. In another embodiment, the modified associated nucleic acids of the array produced using the methods of the invention exhibit substantial resistance to nuclease degradation.

FIELD OF THE INVENTION

The field of this invention is arrays having associated oligonucleotidesand/or polynucleotides, methods of producing such arrays, and usesthereof.

BACKGROUND OF THE INVENTION

Arrays of binding agents, such as oligonucleotides and polynucleotides,have become an increasingly important tool in the biotechnology industryand related fields. These arrays, in which a plurality of binding agentsare deposited onto a solid support surface in the form of an array orpattern, find use in a variety of applications, including drugscreening, nucleic acid sequencing, mutation analysis, and the like. Oneimportant use of arrays is in the analysis of differential geneexpression, where the expression of genes in different cells, normally acell of interest and a control, is compared and any discrepancies inexpression are identified. In such assays, the presence of discrepanciesindicates a difference in the classes of genes expressed in the cellsbeing compared.

In methods of differential gene expression, arrays find use by servingas a substrate with associated binding fragments such asoligonucleotides. Nucleic acid sequences are obtained from analogouscells, tissues or organs of a healthy and diseased organism, andhybridized to the immobilized set of binding fragments associated withthe array. Differences between the resultant hybridization patterns arethen detected and related to differences in gene expression in the twosources.

A variety of different array technologies have been developed in orderto meet the growing need of the biotechnology industry. Despite the widevariety of array technologies currently in preparation or available onthe market, there is a continued need to identify new array devices tomeet the needs of specific applications. Of particular interest arearrays which provide increased binding affinity, because these allow theuse of shorter binding fragments and fewer bound fragments can be usedto obtain the results currently available with conventional technology.Also of interest is the development of an array capable of providinghigh throughput analysis of differential gene expression, where thearray itself is reusable. Such an array is needed for a number ofreasons such as decreasing experimental variability, confirming results,and for decreasing costs of such analysis.

SUMMARY OF THE INVENTION

The present invention provides methods of producing arrays havingassociated oligonucleotide and/or polynucleotides with modifiedstructures (e.g., 1′, 2′, 3′, 5′ modifications, modifications to theribose oxygen, and 2′-substitutions), methods of making such arrays,assays for using such arrays, and kits containing such arrays. Themodifications described herein provide numerous advantages, including ahigher binding affinity for complementary nucleic acids, acidresistance, and/or nuclease resistance. The invention comprises an arraydevice comprised of a support surface and polymer molecules bound to thesupport surface. The polymer molecules are not naturally occurringoligonucleotides or polynucleotides, but rather have modified backboneswith bases attached in the desired sequential positioning and thedesired spacing between the bases. The backbone is preferably modifiedto obtain improved results compared to natural oligonucleotides orpolynucleotides including (1) higher binding affinity; (2) greater acidresistance; (3) greater resistance to enzymatic degradation; and/or (4)overall better performance and reusability.

In one embodiment, the modified associated oligonucleotides and/orpolynucleotides of the invention provide additional binding affinitywith respect to corresponding, unmodified oligonucleotides having thesame sequence. The binding affinity is preferably increased by amodification at the 2′ site of the sugar group, e.g., a 2′-F or a 2′-ORmodification such as 2′-O-methyl or 2′-O-methoxyethoxy. Alternatively orin combination, the binding affinity can be increased by modification inthe 3′ linkage group, e.g., phosphoramidate linkages, or a modificationreplacing the oxygen with a carbon.

In another embodiment, the modified associated oligonucleotides and/orpolynucleotides of the array exhibit substantial acid resistance,allowing the arrays to be treated with low pH solutions. This allows thearray to be exposed to low pH in order to remove any bound nucleic acidsthat are not modified, e.g., bound test nucleic acids.

It is thus an object of the present invention to provide arrays havingassociated chemically modified oligonucleotides and/or polynucleotidescharacterized by substantial acid resistance. Such arrays may be exposedto low pH environments to facilitate clearance from the array of thetest binding agents.

In yet another embodiment, the modified associated oligonucleotidesand/or polynucleotides of the array exhibit substantial resistance tonuclease degradation. These molecules preferably have an end-blockinggroup that confers nuclease resistance to the molecule, e.g., a butanolor butyl group.

It is thus an object of the invention to provide arrays havingassociated chemically modified oligonucleotides and/or polynucleotidesto confer substantial nuclease resistance. Nucleases can be used todigest the test substrate binding agent, freeing the associated bindingagents for further use. The chemical modification may be on the 5′ endfor oligonucleotides and/or polynucleotides attached to a substrate atthe 3′ end, or alternatively the chemical modification may be on the 3′end for oligonucleotides and/or polynucleotides attached to a substrateat the 5′ end. The associated oligonucleotides and/or polynucleotidesremain unaffected as to the binding capacity of the associatedoligonucleotides.

These arrays also offer the significant advantage that the individualchip can be tested for efficacy and/or quality prior to use with a testsample, which is particularly helpful if the amount of test sample islimited or if the array is being used as a medical device and mustcomply with FDA quality control requirements.

The present invention further provides an assay using the arrays of theinvention to determine physiological responses such as gene expression,where the response is determined by the hybridization pattern of thearray after exposure to test samples. The test samples may be mRNA,cDNA, whole cell extracts, and the like.

It is an advantage of the associated modified oligonucleotides and/orpolynucleotides of the arrays of the invention that the chemicalmodifications enhance the chemical binding interactions, e.g., increasebinding affinity over standard Watson-Crick base pairing withcomplementary oligonucleotides and/or polynucleotides, particularly whenbinding to mRNA.

It is another advantage that the modified oligonucleotides and/orpolynucleotides of the array may be synthesized to have approximatelythe same T_(m), by varying the length of the nucleic acids in eachcomposition.

It is another advantage that modified oligonucleotides and/orpolynucleotides of the invention hybridize more tightly withcomplementary RNA sequences than natural DNA oligonucleotides, allowingthe use of shorter binding fragments (e.g. one or more oligonucleotidesin lieu of a complete cDNA).

It is an advantage of the associated modified oligonucleotides and/orpolynucleotides of the invention that the acid stable modificationsconfer an improved stability on the modified oligonucleotides and/orpolynucleotides in an acidic environment (e.g., as low as pH of 1 to 2).

It is another advantage of the associated oligonucleotides and/orpolynucleotides of the invention that they bind with specificity to testnucleic acids.

It is an object of the invention that the oligonucleotides and/orpolynucleotides can be used in a variety of array applications, such asidentification of new genes, determination of expression levels,diagnosis of disease, and the like.

These and other objects, advantages, and features of the invention willbecome apparent to those skilled in the art upon reading the details ofthe oligonucleotides and/or polynucleotides and uses thereof as morefully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-7 illustrate the chemical structure of exemplary modificationsthat result in acid stability.

FIGS. 8-9 illustrate the chemical structure of end-blocked, acid stablemolecules used in the invention.

FIG. 10 illustrates other potential modifications that may be used inthe present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is to be understood that this invention is not limited to theparticular methodology, support surfaces, materials and modificationsdescribed and as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anoligonucleotide” may include a plurality of oligonucleotide moleculesand equivalents thereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned are incorporated herein by reference for thepurpose of describing and disclosing, for example, materials,constructs, and methodologies that are described in the publicationswhich might be used in connection with the presently describedinvention. The publications discussed above and throughout the text areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention.

DEFINITIONS

The terms “nucleic acid” and “nucleic acid molecule” as usedinterchangeably herein, refer to a molecule comprised of one or morenucleotides, i.e., ribonucleotides, deoxyribonucleotides, or both. Theterm includes monomers and polymers of ribonucleotides anddeoxyribonucleotides, with the ribonucleotides and/ordeoxyribonucleotides being connected together, in the case of thepolymers, via 5′ to 3′ linkages. However, linkages may include any ofthe linkages known in the nucleic acid synthesis art including, forexample, nucleic acids comprising 5′ to 2′ linkages. The nucleotidesused in the nucleic acid molecule may be naturally occurring or may besynthetically produced analogues that are capable of forming base-pairrelationships with naturally occurring base pairs. Examples ofnon-naturally occurring bases that are capable of forming base-pairingrelationships include, but are not limited to, aza and deaza pyrimidineanalogues, aza and deaza purine analogues, and other heterocyclic baseanalogues, wherein one or more of the carbon and nitrogen atoms of thepurine and pyrimidine rings have been substituted by heteroatoms, e.g.,oxygen, sulfur, selenium, phosphorus, and the like.

The term “oligonucleotide” as used herein refers to a nucleic acidmolecule comprising from about 2 to about 300 nucleotides.Oligonucleotides for use in the present invention are preferably from80-200, more preferably from 100-150 in length.

The term “polynucleotide” as used herein refers to nucleic acidmolecules comprising a plurality of nucleotide monomers including butnot limited to nucleic acid molecules comprising over 200 nucleotides.

The terms “modified oligonucleotide” and “modified polynucleotide” asused herein refers to oligonucleotides and/or polynucleotides with oneor more chemical modifications at the molecular level of the naturalmolecular structures of all or any of the bases, sugar moieties,internucleoside phosphate linkages, as well as to molecules having addedsubstituents, such as diamines, cholesterol or other lipophilic groups,or a combination of modifications at these sites. The internucleosidephosphate linkages can be phosphodiester, phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, phosphorothioate, methylphosphonate, phosphorodithioate,bridged phosphorothioate and/or sulfone internucleotide linkages, or3′-3′, 5′-2′ or 5′-5′ linkages, and combinations of such similarlinkages (to produce mixed backbone modified oligonucleotides). Themodifications can be internal (single or repeated) or at the end(s) ofthe oligonucleotide molecule, and can include additions to the moleculeof the internucleoside phosphate linkages, such as cholesteryl, diaminecompounds with varying numbers of carbon residues between amino groupsand terminal ribose, and deoxyribose and phosphate modifications whichcleave or cross-link to the opposite chains or to associated enzymes orother proteins. Electrophilic groups such as ribose-dialdehyde couldcovalently link with an epsilon amino group of the lysyl-residue of sucha protein. A nucleophilic group such as n-ethylmaleimide tethered to anoligomer could covalently attach to the 5′ end of an mRNA or to anotherelectrophilic site. The terms “modified oligonucleotides” and “modifiedpolynucleotides” also include oligonucleotides and/or polynucleotidescomprising modifications to the sugar moieties (e.g., 2′-substitutedribonucleotides or deoxyribonucleotide monomers), any of which areconnected together via 5′ to 3′ linkages. Modified oligonucleotides mayalso be comprised of PNA or morpholino modified backbones where targetspecificity of the sequence is maintained. A modified oligonucleotide ofthe invention (1) does not have the structure of a naturally occurringoligonucleotide and (2) will hybridize to a natural oligonucleotidemRNA, or cDNA. Further, the modification preferably provides (3) higherbinding affinity, (4) greater acid resistance, and (5) better stabilityagainst digestion with enzymes as compared to a natural oligonucleotide.

The term “oligonucleotide backbone” as used herein refers to thestructure of the chemical moiety linking nucleotides in a molecule. Theinvention preferably comprises a backbone which is different from anaturally occurring backbone and is further characterized by (1) holdingbases in correct sequential order and (2) holding bases a correctdistance between each other to allow a natural oligonucleotide tohybridize to it. This may include structures formed from any and allmeans of chemically linking nucleotides. A modified backbone as usedherein includes modifications (relative to natural linkages) to thechemical linkage between nucleotides, as well as other modificationsthat may be used to enhance stability and affinity, such asmodifications to the sugar structure. For example an α-anomer ofdeoxyribose may be used, where the base is inverted with respect to thenatural β-anomer. In a preferred embodiment, the 2′-H or 2′-OH of thesugar group (for RNA and DNA, respectively) may be altered to 2′-O-alkylor 2′-O-alkyl-n(O-alkyl), which provides resistance to degradationwithout comprising affinity.

The term “end-blocked” as used herein refers to an oligonucleotide witha chemical modification at the molecular level that prevents thedegradation of selected nucleotides, e.g., by nuclease action. Thischemical modification is positioned such that it protects the integralportion of the oligonucleotide, for example the region of theoligonucleotide that is targeted for hybridization (i.e., the testsequence of the oligonucleotide). An end block may be a 3′ end block ora 5′ end block. For example, a 3′ end block may be at the 3′-mostposition of the molecule, or it may be internal to the 3′ ends, providedit is 3′ of the integral sequences of the oligonucleotide.

The term “substantially nuclease resistant” refers to oligonucleotidesthat are resistant to nuclease degradation as compared to naturallyoccurring or unmodified oligonucleotides. Modified oligonucleotides ofthe invention are at least 1.25 times more resistant to nucleasedegradation than their unmodified counterpart, more preferably at least2 times more resistant, even more preferably at least 5 times moreresistant, and most preferably at least 10 times more resistant thantheir unmodified counterpart. Such substantially nuclease resistantoligonucleotides include, but are not limited to, oligonucleotides withmodified backbones such as phosphorothioates, methylphosphonates,ethylphosphotriesters, 2′-O-methylphosphorothioates,2′-O-methyl-p-ethoxy ribonucleotides, 2′-O-alkyls,2′-O-alkyl-n(O-alkyl), 3′-O-alkyls, 3′-O-alkyl-n(O-alkyl), 3′-O-methylribonucleotides, 2′-fluoros, 2′-deoxy-erythropentofuranosyls,2′-O-methyl ribonucleosides, methyl carbamates, methyl carbonates,inverted bases (e.g., inverted T's), or chimeric versions of thesebackbones.

The term “substantially acid resistant” as used herein refers tooligonucleotides that are resistant to acid degradation as compared tounmodified oligonucleotides. Typically, the relative acid resistance ofan oligonucleotide will be measured by comparing the percent degradationof a resistant oligonucleotide with the percent degradation of itsunmodified counterpart (i.e., a corresponding oligonucleotide with“normal” backbone, bases, and phosphodiester linkages). Anoligonucleotide that is acid resistant is preferably at least 1.5 timesmore resistant to acid degradation, at least 2 times more resistant,even more preferably at least 5 times more resistant, and mostpreferably at least 10 times more resistant than their unmodifiedcounterpart.

The term “alkyl” as used herein refers to a branched or unbranchedsaturated hyrdrocarbon chain containing 1-6 carbon atoms, such asmethyl, ethyl, propyl, tert-butyl, n-hexyl and the like.

The term “array type” refers to the type of gene represented on thearray by the associated test oligonucleotides, where the type of genethat is represented on the array is dependent on the intended purpose ofthe array, e.g., to monitor expression of key human genes, to monitorexpression of known oncogenes, etc., i.e., the use for which the arrayis designed. As such, all of the test oligonucleotides on a given arraycorrespond to the same type or category or group of genes. Genes areconsidered to be of the same type if they share some common linkingcharacteristics, such as: species of origin, e.g., human, mouse, rat,etc.; tissue or cell type of origin, e.g., muscle, neural, dermal,organ, etc.; disease state, e.g., cancer; functions, e.g., proteinkinases, tumor supressors and the like; participation in the same normalbiological process, e.g. apoptosis, signal transduction, cell cycleregulation, proliferation, differentiation etc.; and the like. Forexample, one array type is a “cancer array” in which each of the“unique” associated test oligonucleotides correspond to a geneassociated with a cancer disease state. Likewise, a “human array” may bean array of test oligonucleotides corresponding to unique tightlyregulated human genes. Similarly, an “apoptosis array” may be an arraytype in which the associated test oligonucleotides correspond to uniquegenes associated with apoptosis.

The terms “associated oligonucleotide,” “associated polynucleotide” and“substrate oligonucleotide” and the like refer to the oligonucleotide orpolynucleotide composition that makes up each of the samples associatedto the array. Thus, the term “associated oligonucleotide” includesoligonucleotide compositions of unique sequences and/or control orcalibrating sequences (e.g., oligonucleotides corresponding tohousekeeping genes). The oligonucleotide and/or polynucleotidecompositions are preferably comprised of single stranded nucleic acid,where all of the nucleic acids in a sample composition may be identicalto each other. Alternatively, there may be nucleic acids having two ormore sequences in each composition, for example two differentoligonucleotides that are separate but complementary to each other.

The Invention in General

Arrays having associated oligonucleotides and/or polynucleotides withmodified backbone structures, such as oligonucleotides with 2′-O-alkyland 2′-O-alkyl-n(O-alkyl) sugar moieties, changes in the ribose oxygen,5′ linkage modifications, and/or 3′ linkage modifications, are provided.Modified oligonucleotides and polynucleotides of the invention also maybe acid resistant and/or exonuclease resistant to further decrease thesensitivity of the oligonucleotide molecule. In one embodiment, anexonuclease resistant block is added to the 3′ or the 5′ end of theoligonucleotide or polynucleotide depending on the attachment of thenucleic acid to the substrate. The resulting modified oligonucleotidesand/or polynucleotides of the invention bind tightly to their RNA or DNAtargets.

Modified oligonucleotides and/or polynucleotides of the inventionpreferably have an increased binding affinity over their non-modifiedRNA or DNA counterparts. This binding affinity can be determined usingT_(m) assays such as those described in L. L. Cummins et al., NucleicAcids Research 23:2019-2024 (1995). Typically, the T_(m) of anoligonucleotide binding to RNA will increase approximately 1° C. foreach 2′-O-methyl substitution in a molecule, and the T_(m) increaseseven more for 2′-O-propyl and 2′-F substitutions. Thus, in oneembodiment, the T_(m) of the modified oligonucleotide bound to RNA is2-15° C., and even more preferably 8-10° C. higher than thecorresponding non-modified DNA oligonucleotide.

The modified oligonucleotides and/or polynucleotides of the array may besynthesized to have approximately the same T_(m), by varying the lengthof the nucleic acids in each composition. Thus, an oligonucleotide withan A-T rich sequence would be designed to be longer than anoligonucleotide with a G-C rich sequence to provide approximately thesame T_(m). The T_(m) of each of the compositions on an array can beheld relatively constant by providing lengths of oligonucleotides andpolynucleotides based on the binding affinity of the base sequence.

Acid stable associated oligonucleotides and/or polynucleotides of theinvention are stable when exposed to a pH of 1-2, while their bindingpartners are not. This allows an array having associated acid stableoligonucleotides and/or polynucleotides to be exposed to a first sample,treated with an acidic solution applied in any of several possibleprotocols to free the array from the first binding partner, and reusedwith a second sample. Direct comparison of two different samples ofbinding partners using a single array has the advantage of limitingpotential experimental variation present when comparing multiple arrays.Performing the experiment with the same sample on the same array allowsa confirmation of the results obtained in the first instance, thuseffectively confirming results without having variation in the arraycomposition.

Similarly, associated end-blocked oligonucleotides and/orpolynucleotides display a resistance to nucleases, allowing the arraysto be exposed to DNA nucleases to free the array from a sample ofbinding partners. An array of the invention having nuclease resistantassociated oligonucleotides can be treated with an appropriate nucleaseand reused with a different or the same sample.

The arrays of the present invention encompassassociated,oligonucleotides chemically modified to be acid stable from apH of 0.01 to 7.0, and more preferably acid stable in a pH of 1.0 to4.0, allowing such molecules to retain their structural integrities inacidic environments. Although any 2′-modified oligonucleotide may beused in the present invention, in a preferred embodiment theoligonucleotides of the invention are 2′-O-alkyl and2′-O-alkyl-n(O-alkyl) oligonucleotides which, unlike unsubstitutedphosphodiester or phosphorothioate DNA or RNA, exhibit significant acidresistance in solutions with pH as low as 0-1 even at 37° C. Acidstability of this first component coupled with the introduction of 3′and/or 5′ acid stable, exonuclease resistant ends, confers severalunique properties on 2′-O-alkyl and 2′-O-alkyl-n(O-alkyl)oligonucleotides. These low toxicity, highly specific, acid stable,end-blocked 2′-O-alkyl and 2′-O-alkyl-n(O-alkyl) oligonucleotidesrepresent a novel and improved oligonucleotide structure for use inarray technologies.

Typically, the relative nuclease resistance of a oligonucleotide can bemeasured by comparing the percent digestion of a resistantoligonucleotide with the percent digestion of its unmodified counterpart(i.e., a corresponding oligonucleotide with “normal” backbone, bases,and phosphodiester linkage). Percent degradation may be determined byusing analytical HPLC to assess the loss of full lengtholigonucleotides, or by any other suitable methods (e.g., by visualizingthe products on a sequencing gel using staining, autoradiography,fluorescence, etc., or measuring a shift in optical density).Degradation is generally measured as a function of time.

Comparison between unmodified and modified oligonucleotides can be madeby ratioing the percentage of intact modified oligonucleotide to thepercentage of intact unmodified oligonucleotide. For example, if, after15 minutes of exposure to a nuclease, 25% (i.e., 75% degraded) of anunmodified oligonucleotide is intact, and 50% (i.e., 50% degraded) of amodified oligonucleotide is intact, the modified oligonucleotide is saidto be 2 times (50% divided by 25%) more resistant to nucleasedegradation than is the unmodified oligonucleotide. Generally, asubstantially nuclease resistant oligonucleotide will be at least about1.25 times more resistant to nuclease degradation than an unmodifiedoligonucleotide with a corresponding sequence, typically at least about1.5 times more resistant, preferably about 1.75 times more resistant,and more preferably at least about 10 times more resistant after 15minutes of nuclease exposure.

Percent acid degradation may be determined by using analytical HPLC toassess the loss of full length oligonucleotides, or by any othersuitable methods (e.g., by visualizing the products on a sequencing gelusing staining, autoradiography, fluorescence, etc., or measuring ashift in optical density). Degradation is generally measured as afunction of time.

Comparison between unmodified and modified oligonucleotides can be madeby ratioing the percentage of intact modified oligonucleotide to thepercentage of intact unmodified oligonucleotide. For example, if, after30 minutes of exposure to a low pH environment, 25% (i.e., 75% degraded)of an unmodified oligonucleotide is intact, and 50% (i.e., 50% degraded)of a modified oligonucleotide is intact, the modified oligonucleotide issaid to be 2 times (50% divided by 25%) more resistant to nucleasedegradation than is the unmodified oligonucleotide. Generally,substantially “acid resistant” oligonucleotides will be at least about1.25 times more resistant to acid degradation than an unmodifiedoligonucleotide with a corresponding sequence, typically at least about1.5 times more resistant, preferably about 1.75 more resistant, morepreferably at least 5 times more resistant and even more preferably atleast about 10 times more resistant after 30 minutes of exposure at 37°C. to a pH of about 1.5 to about 4.5.

In a preferred embodiment, the end-blocked oligonucleotides of thecompositions and methods of the invention are substantially nucleaseresistant, substantially acid resistant, and preferably, bothsubstantially nuclease resistant and substantially acid resistant. Thisembodiment includes oligonucleotides completely or partially derivatizedby one or more linkages from the group comprised of phosphorothioatelinkages, 2′-O-methyl-phosphodiesters, 2′-O-alkyl, 2′-O-ethyl,2′-O-propyl, 2′-O-butyl, 2′-O-alkyl-n(O-alkyl), 2′-methoxyethoxy,2′-fluoro, 2′-deoxy-erythropentofuranosyl, 3′-O-methyl, p-isopropyloligonucleotides, phosphodiester, 2′-O(CH₂CH₂O)_(x)CH₃, butyne,phosphotriester, phosphoramidate, propargyl, siloxane, carbonate,carboxymethylester, methoxyethoxy, acetamidate, carbamate, thioether,bridged phosphoramidate, bridged methylene phosphonate,methylphosphonate, phosphorodithioate, bridged phosphorothioate and/orsulfone internucleotide linkages, or 3′-3′ or 5′-5′ or 5′-2′ linkages,and combinations of such similar linkages (to produce mixed backbonemodified oligonucleotides), and any other backbone modifications.

Exemplary modifications that result in acid stability can be seen inFIGS. 1-6. End-blocked acid stable molecules are illustrated in FIGS.7-8. Other modifications that may be of use in the present invention areillustrated. See “The Medicinal Chemistry of Oligonucleotides” inMedical Intelligence Unit: Therapeutic Applications of Oligonucleotides(1995) pp. 85-108; and Mesmaeker et al., Acc. Chem. Res., 28:366-374(1995).

This embodiment also includes other modifications that render theoligonucleotides and/or polynucleotides substantially resistant tonuclease activity. Methods of rendering an oligonucleotide nucleaseresistant include, but are not limited to, covalently modifying thepurine or pyrimidine bases that comprise the oligonucleotide. Forexample, bases may be methylated, hydroxymethylated, or otherwisesubstituted (e.g., glycosylated) such that the oligonucleotidescomprising the modified bases are rendered substantially nucleaseresistant.

In a preferred embodiment, the oligonucleotide and/or polynucleotidewill have a backbone substantially resistant to acid degradation,exonuclease digestion, and endonuclease digestion. In the most preferredembodiment an oligonucleotide is uniformly modified with 2′-O-alkyl or2′-O-alkyl-n(O-alkyl) groups, i.e., every base of the oligonucleotide isa 2′-O-alkyl or 2′-O-alkyl-n(O-alkyl) modified base.

In another embodiment, the associated oligonucleotides and/orpolynucleotides of the current invention are used for diagnosticpurposes. For example, oligonucleotides of the current invention may beused to detect complementary oligonucleotides by contacting anoligonucleotide of the invention with an oligonucleotide sample underconditions that allow for the hybridization of the oligonucleotide ofthe invention to any complementary oligonucleotide present in thesample, and detecting such hybridization.

Oligonucleotides with a range of nuclease-resistant backbones wereevaluated. As a result, a preferred embodiment of the present inventionis an end-blocked oligonucleotide with the chemical backbone structureof 5′-butanol-2′-O-alkyl RNA-butanol-3′ or 2′-O-alkyl-O-alkyl. Aparticularly preferred embodiment of the present invention is anoligonucleotide with the chemical backbone structure of5′-butanol-2′-O-methyl RNA-butanol-3′,5′-butanol-2′-O-alkyl-O-alkylRNA-butanol-3′ or 2′-O-alkyl-O-alkyl RNA. The end-blocking group on oneend of the oligonucleotide may not be needed, depending on the manner ofassociation with the substrate, as will be apparent to one skilled inthe art upon reading the present disclosure.

Associated Oligonucleotide and Polynucleotide Compositions of the Arrays

Each associated oligonucleotide and/or polynucleotide composition of thepattern present on the surface of the substrate is preferably made up ofa set of unique nucleic acids, and preferably a unique oligonucleotidecomposition. By “unique composition” is meant a collection or populationof single stranded oligonucleotides capable of participating in ahybridization event under appropriate hybridization conditions, whereeach of the individual oligonucleotides may be the same—have the samenucleotide sequence—or different sequences, for example theoligonucleotide composition may consist of two differentoligonucleotides that are complementary to each other (i.e., the twodifferent oligonucleotides are complementary but physically separated soas to be single stranded, i.e., not hybridized to each other). In manyembodiments, the oligonucleotide compositions will comprise twocomplementary, single stranded oligonucleotides.

In those compositions having unique oligonucleotides, the sequence ofthe oligonucleotides are chosen in view of the type and the intended useof the array on which they are present. The unique oligonucleotides arepreferably chosen so that each distinct unique oligonucleotide does notcross-hybridize with any other distinct unique oligonucleotide on thearray, i.e., the oligonucleotide will not cross-hybridize to any otheroligonucleotide compositions that correspond to a different gene fallingwithin the broad category or type of genes represented on the arrayunder appropriate conditions. As such, the nucleotide sequence of eachunique oligonucleotide of a composition will have less than 90%homology, usually less than 85% homology, and more usually less than 80%homology with any other different associated oligonucleotide compositionof the array, where homology is determined by sequence analysiscomparison using the FASTA program using default settings. The sequenceof unique associated oligonucleotides in the compositions are notconserved sequences found in a number of different genes (at least two),where a conserved sequence is defined as a stretch of from about 4 toabout 80 nucleotides which have at least about 90% sequence identity,where sequence identity is measured as above. The associatedoligonucleotide will generally have a length of from about 80 to about300 nucleotides, usually from 100 to about 200 nucleotides. The lengthof the nucleic acid can be chosen to best provide binding to the testsequence.

Although in a preferred embodiment the associated modifiedoligonucleotide composition will not cross-hybridize with any otherassociated oligonucleotides on the array under standard hybridizationconditions, associated oligonucleotides and hybridization conditions canbe altered to allow binding to multiple associated oligonucleotidecompositions. For example, in determining the relatedness of a sample tooligonucleotides representing different members of a class of proteins,the oligonucleotide sequences may be more similar and/or less stringenthybridization conditions may be used.

Chemical Modifications of Oligonucleotides and/or Polynucleotides of theInvention

The oligonucleotides and/or polynucleotides of the invention may containany modification that confers on the molecules greater binding withother nucleic acids, that increases the acid stability and/or increasesthe nuclease stability of the molecule. This includes oligonucleotidesand/or polynucleotides completely derivatized by phosphorothioatelinkages, 2′-O-methylphosphodiesters, 2′-O-alkyl, 2′-O-alkyl-n(O-alkyl),2′-fluoro, 2′-deoxy-erythropentofuranosyl, 3′-O-methylphosphodiesters,p-ethoxy oligonucleotides, p-isopropyl oligonucleotides,phosphoramidates, phosphoroamidites, chimeric linkages, carbonates,amines, formacetals, silyls and siloxys, sulfonates, hydrocarbon,amides, ureas and any other backbone modifications, as well as othermodifications, which render the oligonucleotides and/or polynucleotidessubstantially resistant to endogenous nuclease activity. The nucleotidesin each oligonucleotide and/or polynucleotide may each contain the samemodifications, may contain combinations of these modifications, or maycombine these modifications with phosphodiester linkages. Additionalmethods of rendering oligonucleotides and/or polynucleotides nucleaseresistant include, but are not limited to, covalently modifying thepurine or pyrimidine bases that comprise the oligonucleotide. Forexample, bases may be methylated, hydroxymethylated, or otherwisesubstituted (e.g., glycosylated) such that the oligonucleotides and/orpolynucleotides comprising the modified bases are rendered substantiallyacid and nuclease resistant.

The ring structure of the ribose group of the nucleotides in themodified oligonucleotide and/or polynucleotide may also have an oxygenin the ring structure substituted with N—H, N—R, S and/or methylene.

Although 2′-O-alkyl substituted oligonucleotides and/or polynucleotidesexhibit marked acid stability and endonuclease resistance, they aresensitive to 3′ exonucleases. In order to enhance the exonucleaseresistance of 2′-O-alkyl substituted oligonucleotides and/orpolynucleotides, the 3′ or 5′ and 3′ ends of the ribooligonucleotidesequence are preferably attached to an exonuclease blocking function.For example, one or more phosphorothioate nucleotides can be placed ateither end of the oligoribonucleotide. Additionally, one or moreinverted bases can be placed on either end of the oligoribonucleotide,or one or more alkyls, e.g., butanol-substituted nucleotides or chemicalgroups, can be placed on one or more ends of the oligoribonucleotide.Accordingly, a preferred embodiment of the present invention is anoligonucleotide comprising a oligonucleotide having the followingstructure:A-B—Cwherein “B” is a 2′-O-alkyl or 2′-O-alkyl-n(O-alkyl) oligoribonucleotidebetween about 2 and about 300 bases in length, and “A” and “C” arerespective 5′ and 3′ end blocking groups (e.g., one or morephosphorothioate nucleotides (but typically fewer than six), invertedbase linkages, or alkyl, alkenyl, or alkynl groups or substitutednucleotides or 2′-O-alkyl-n(O-alkyl)). A partial list of blocking groupsincludes inverted bases, dideoxynucleotides, methylphosphates, alkylgroups, aryl groups, cordycepin, cytosine arabanoside, 2′-methoxy,ethoxy nucleotides, phosphoramidates, a peptide linkage, dinitrophenylgroup, 2′- or 3′-O-methyl bases with phosphorothioate linkages,3′-O-methyl bases, fluorescein, cholesterol, biotin, acridine,rhodamine, psoralen, glyceryl, methyl phosphonates, butanol, butyl,hexanol, and 3′-O-alkyls. An enzyme-resistant butanol preferably has thestructure OH—CH₂CH₂CH₂CH₂ (4-hydroxybutyl) which is also referred to asa C4 spacer.Oligonucleotide and Polynucleotide Synthesis

Oligonucleotides can be synthesized on commercially purchased DNAsynthesizers from <1 uM to >1 mM scales using standard phosphoramiditechemistry and methods that are well known in the art, such as, forexample, those disclosed in Stec et al., J. Am. Chem. Soc. 106:6077-6089(1984), Stec et al., J. Org. Chem. 50(20):3908-3913 (1985), Stec et al.,J. Chromatog. 326:263-280 (1985), LaPlanche et al., Nuc. Acid. Res.14(22):9081-9093 (1986), and Fasman, Practical Handbook of Biochemistryand Molecular Biology, 1989, CRC Press, Boca Raton, Fla., hereinincorporated by reference.

Oligonucleotides can be deprotected following phosphoramiditemanufacturer's protocols. Unpurified oligonucleotides may be dried downunder vacuum or precipitated and then dried. Sodium salts ofoligonucleotides can be prepared using the commercially availableDNA-Mate (Barkosigan Inc.) reagents or conventional techniques such as acommercially available exchange resin, e.g., Dowex, or by addition ofsodium salts followed by precipitation, diafiltration, or gelfiltration, etc.

Oligonucleotides to be purified can be chromatographed on commerciallyavailable reverse phase or ion exchange media, e.g., Waters Protein Pak,Pharmacia's Source Q, etc. Peak fractions can be combined and thesamples desalted and concentrated by means of reverse phasechromatography on poly(styrene-divinylbenzene) based columns likeHamilton's PRP, or Polymer Labs PLRP.

Alternatively, ethanol precipitation, diafiltration, or gel filtrationmay be used followed by lyophilization or solvent evaporation undervacuum in commercially available instrumentation such as Savant's SpeedVac. Optionally, small amounts of the oligonucleotides may beelectrophoretically purified using polyacrylamide gels.

Lyophilized or dried-down preparations of oligonucleotides can bedissolved in pyrogen-free, sterile, physiological saline (i.e., 0.85%saline), sterile Sigma water, and filtered through a 0.45 micron Gelmanfilter (or a sterile 0.2 micron pyrogen-free filter). The describedoligonucleotides may be partially or fully substituted with any of abroad variety of chemical groups or linkages including, but not limitedto: phosphoramidates; phosphorothioates; alkyl phosphonates,2′-O-methyls; 2′-modified RNAs; morpholino groups; phosphate esters;propyne groups; or chimerics of any combination of the above groups orother linkages (or analogs thereof).

A variety of standard methods can be used to purify the presentlydescribed oligonucleotides. In brief, the oligonucleotides of thepresent invention can be purified by chromatography on commercialyavailable reverse phase (for example, see the RANIN Instrument Co., Inc.instruction manual for the DYNAMAX®-300A, Pure-DNA reverse-phasecolumns, 1989, or current updates thereof, herein incorporated byreference) or ion exchange media such as Waters' Protein Pak orPharmacia's Source Q (see generally, Warren and Vella, 1994, “Analysisand Purification of Synthetic Nucleic Acids by High-Performance LiquidChromatography”, in Methods in Molecular Biology, vol. 26; Protocols forNucleic Acid Conjugates, S. Agrawal, Ed., Humana Press, Inc., Totowa,N.J.; Aharon et al., 1993, J. Chrom. 698:293-301; and MilliporeTechnical Bulletin, 1992, Antisense DNA: Synthesis, Purification, andAnalysis). Peak fractions can be combined and the samples concentratedand desalted via alcohol (ethanol, butanol, isopropanol, and isomers andmixtures thereof, etc.) precipitation, reverse phase chromatography,diafiltration, or gel filtration.

The modified polynucleotides and oligonucleotides that are associated onthe array may also be produced used established techniques such aspolymerase chain reaction (PCR) and reverse transcription (RT). Thesemethods are similar to those currently known in the art (see e.g., PCRStrategies, Michael A. Innis (Editor), et al. (1995) andPCR:Introduction to Biotechniques Series, C. R. Newton, A. Graham(1997)), and preferably the enzymes used to produce the polynucleotidesor oligonucleotides are optimized for incorporation of modifiednucleotide monomers. Methods of identifying which enzymes are bestsuited for incorporation of nucleotide monomers with specificmodifications (e.g., which enzymes will best incorporate 2′-modifieddNTPs) are well known in the art, and thus one skilled in the art wouldbe able to identify enzymes for use with the present invention basedupon this disclosure. For example, the process directed evolution can beused to unveil mechanisms of both thermal adaptation and incorporationefficiency, and is an effective and efficient approach to identifyingoptimal enzyme activity. Multiple generations of random mutagenesis,recombination and high throughput can be used to create a polymerasethat both incorporates modified nucleotide monomers, e.g., 2′-O-methylsubstituted dNTPs, and remains thermostable at higher temperatures. Seee.g., Zhao H, et al. 12:47-53 (1999).

Other methods of altering catalytic activity include site-directedmutagenesis, codon-level mutagenesis and methods of incorporatingdeletions or insertions into available enzymes. Genomic sequencingprograms may also reveal conserved regions in the enzyme structure andregions of variability between enzymes from closely related species,thus identifying regions of an enzyme that may be altered withoutaffecting the desired activity. It would be well within the skill of onein the art to use such techniques to identify an enzyme with optimalperformance for producing the modified polynucleotides andoligonucleotides of the invention.

Techniques for identification of specific enzymes for production ofpolynucleotides for association on the arrays of the invention aredescribed in Schmidt-Dannert C, et al., Trends Biotechnol. 17:135-6(1999); Moreno-Hagelsieb G, et al., Biol Res.29:127-40 (1996); ColacinoF, et al., Biotechnol Genet Eng Rev. 14:211-77 (1997); Soberon X. NatBiotechnol. 17:539-40 (1999); Arnold F. H., et al., Ann N Y Acad Sci.870:400-3 (1999); and Joo H, et al., Nature 399:670-3 (1999), each ofwhich are incorporated herein by reference to describe such techniquesand enzyme design.

An oligonucleotide or polynucleotide is considered pure when it has beenisolated so as to be substantially free of, inter alia, incompleteproducts produced during the synthesis of the desired oligonucleotide orpolynucleotide. Preferably, a purified oligonucleotide or polynucleotidewill also be substantially free of contaminants which may hinder orotherwise mask the binding activity of the molecule.

ARRAY CONSTRUCTION

The arrays of the subject invention have a plurality of associatedmodified oligonucleotides and/or polynucleotides stably associated witha surface of a solid support, e.g., covalently attached to the surfacewith or without a linker molecule. Each associated sample on the arraycomprises a modified oligonucleotide composition, of known identity,usually of known sequence, as described in greater detail below. Anyconceivable substrate may be employed in the invention.

In the arrays of the invention, the modified oligonucleotidecompositions are stably associated with the surface of a solid support,where the support may be a flexible or rigid solid support. By “stablyassociated” is meant that the sample of associated modifiedoligonucleotides and/or polynucleotides maintain their position relativeto the solid support under hybridization and washing conditions. Assuch, the samples can be non-covalently or covalently stably associatedwith the support surface. Examples of non-covalent association includenon-specific adsorption, binding based on electrostatic interactions(e.g., ion pair interactions), hydrophobic interactions, hydrogenbonding interactions, specific binding through a specific binding pairmember covalently attached to the support surface, and the like.Examples of covalent binding include covalent bonds formed between theoligonucleotides and a functional group present on the surface of therigid support (e.g., —OH), where the functional group may be naturallyoccurring or present as a member of an introduced linking group, asdescribed in greater detail below.

As mentioned above, the array is present on either a flexible or rigidsubstrate. A flexible substrate is capable of being bent, folded orsimilarly manipulated without breakage. Examples of solid materialswhich are flexible solid supports with respect to the present inventioninclude membranes, e.g., nylon, flexible plastic films, and the like. By“rigid” is meant that the support is solid and does not readily bend,i.e., the support is not flexible. As such, the rigid substrates of thesubject arrays are sufficient to provide physical support and structureto the associated oligonucleotides and/or polynucleotides presentthereon under the assay conditions in which the array is employed,particularly under high throughput handling conditions. Furthermore,when the rigid supports of the subject invention are bent, they areprone to breakage.

The substrate may be biological, nonbiological, organic, inorganic, or acombination of any of these, existing as particles, strands,precipitates, gels, sheets, tubing, spheres, containers, capillaries,pads, slices, films, plates, slides, etc. The substrate may have anyconvenient shape, such as a disc, square, sphere, circle, etc.

The substrate is preferably flat but may take on a variety ofalternative surface configurations. For example, the substrate maycontain raised or depressed regions on which the synthesis takes place.The substrate and its surface preferably form a rigid support on whichto carry out the reactions described herein. The substrate and itssurface are also chosen to provide appropriate light-absorbingcharacteristics. For instance, the substrate may be a polymerizedLangmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO₂,SIN₄, modified silicon, or any one of a wide variety of gels or polymerssuch as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride,polystyrene, polycarbonate, or combinations thereof. Other substratematerials will be readily apparent to those of skill in the art uponreview of this disclosure.

In a preferred embodiment the substrate is flat glass or single-crystalsilicon with surface relief features of less than 10 angstroms.According to some embodiments, the surface of the substrate is etchedusing well known techniques to provide for desired surface features. Forexample, by way of the formation of trenches, v-grooves, mesastructures, or the like, the synthesis regions may be more closelyplaced within the focus point of impinging light, be provided withreflective “mirror” structures for maximization of light collection fromfluorescent sources, etc.

Surfaces on the solid substrate will usually, though not always, becomposed of the same material as the substrate. Alternatively, thesurface may be composed of any of a wide variety of materials, forexample, polymers, plastics, resins, polysaccharides, silica orsilica-based materials, carbon, metals, inorganic glasses, membranes, orany of the above-listed substrate materials. In some embodiments thesurface may provide for the use of caged binding members which areattached firmly to the surface of the substrate. Preferably, the surfacewill contain reactive groups, which could be carboxyl, amino, hydroxyl,or the like. Most preferably, the surface will be optically transparentand will have surface Si—OH functionalities, such as are found on silicasurfaces.

The surface of the substrate is preferably provided with a layer oflinker molecules, although it will be understood that the linkermolecules are not required elements of the invention. The linkermolecules are preferably of sufficient length to permit modifiedoligonucleotides and/or polynucleotides of the invention and on asubstrate to hybridize to natural nucleic acid molecules and to interactfreely with molecules exposed to the substrate. The linker moleculesshould be 6-50 atoms long to provide sufficient exposure. The linkermolecules may also be, for example, aryl acetylene, ethylene glycololigomers containing 2-10 monomer units, diamines, diacids, amino acids,or combinations thereof. Other linker molecules which can bind tomodified oligonucleotides of the invention may be used in light of thisdisclosure.

The linker molecules can be attached to the substrate via carbon-carbonbonds using, for example, (poly)trifluorochloroethylene surfaces, orpreferably, by siloxane bonds (using, for example, glass or siliconoxide surfaces). Siloxane bonds with the surface of the substrate may beformed in one embodiment via reactions of linker molecules bearingtrichlorosilyl groups. The linker molecules may optionally be attachedin an ordered array, i.e., as parts of the head groups in a polymerizedLangmuir Blodgett film. In alternative embodiments, the linker moleculesare adsorbed to the surface of the substrate.

In one embodiment of the present invention, the linker molecules andmodified nucleotides used herein are provided with a functional group towhich is bound a protective group. Preferably, the protective group ison the distal or terminal end of the linker molecule opposite thesubstrate. The protective group may be either a negative protectivegroup (i.e., the protective group renders the linker molecules lessreactive with a monomer upon exposure) or a positive protective group(i.e., the protective group renders the linker molecules more reactivewith a monomer upon exposure). In the case of negative protective groupsan additional step of reactivation will be required. In someembodiments, this will be done by heating. The protective group on thelinker molecules may be selected from a wide variety of positivelight-reactive groups preferably including nitro aromatic compounds suchas o-nitrobenzyl derivatives or benzylsulfonyl. In a preferredembodiment, 6-nitroveratryloxycarbonyl (NVOC), 2-nitrobenzyloxycarbonyl(NBOC) or α,α-dimethyl-dimethoxybenzyloxycarbonyl (DDZ) is used.Photoremovable protective groups are described in, for example,Patchornik, J. Am. Chem. Soc. (1970) 92:6333 and Amit et al., J. Org.Chem. (1974) 39:192, both of which are incorporated herein by reference.

The substrate, the region for attachment of an individualoligonucleotide group could be of any size or shape. For example,squares, ellipsoids, rectangles, triangles, circles, or portionsthereof, along with irregular geometric shapes, may be utilized.Duplicate synthesis regions may also be applied to a single substratefor purposes of redundancy. The regions on the substrate can have asurface area of between about 1 cm² and 10⁻¹⁰ cm². Preferably, theregions have areas of less than about 10⁻¹ to 10⁻⁷ cm², more preferablyless than 10⁻³ to 10⁻⁶ cm², and even more preferably less than 10⁻⁵ cm².

A single substrate supports more than about 10 different oligonucleotideand/or polynucleotide compositions and preferably more than about 100different oligonucleotide and/or polynucleotide compositions, althoughin some embodiments more than about 10³, 10⁴, 10⁵, 10⁶, 10⁷, or 10⁸different compositions are provided on a substrate. Of course, within aregion of the substrate in which a modified oligonucleotide orpolynucleotide is attached, it is preferred that the modifiednucleotides be substantially pure. In preferred embodiments, regions ofthe substrate contain oligonucleotides or polynucleotides which are atleast about 50%, preferably 80%, more preferably 90%, and even morepreferably, 95% pure. Oligonucleotides or polynucleotides having severalsequences can be intentionally provided within a single region so as toprovide an initial screening for biological activity, after whichmaterials within regions exhibiting significant binding are furtherevaluated. In a preferred embodiment, each region will contain asubstantially pure modified oligonucleotide or polynucleotidecomposition having a single sequence.

The substrates of the arrays of the invention comprise at least onesurface on which the pattern of associated oligonucleotides and/orpolynucleotides is present, where the surface may be smooth,substantially planar, or have irregularities, such as depressions orelevations. The surface on which the pattern of associated nucleic acidspresent may be modified with one or more different layers of compoundsthat serve to modify the properties of the surface in a desirablemanner. Such modification layers, when present, will generally range inthickness from a monomolecular thickness to about 1 mm, usually from amonomolecular thickness to about 0.1 mm and more usually from amonomolecular thickness to about 0.001 mm. Modification layers ofinterest include: inorganic and organic layers such as metals, metaloxides, polymers, small organic molecules and the like.

The amount of modified oligonucleotide or polynucleotide present in eachcomposition will be sufficient to provide for adequate hybridization anddetection of nucleic acids during the assay in which the array isemployed. Generally, the amount of oligonucleotide or polynucleotide ineach composition will be at least about 0.1 ng, usually at least about0.5 ng and more usually at least about 1 ng, where the amount may be ashigh as 1000 ng or higher, but will usually not exceed about 20 ng andmore usually will not exceed about 10 ng. The copy number of eacholigonucleotide or polynucleotide in a composition will be sufficient toprovide enough hybridization sites to yield a detectable signal, andwill generally range from about 0.01 fmol to 50 fmol, usually from about0.05 fmol to 20 fmol and more usually from about 0.1 fmol to 5 fmol.Where the composition has an overall circular dimension, the diameter ofthe sample will generally range from about 10 to 5,000 μm, usually fromabout 20 to 2,000 μm and more usually from about 50 to 1000 μm.

Control composition may be present on the array including compositionscomprising oligonucleotides or polynucleotides corresponding to genomicDNA, housekeeping genes, negative and positive control genes, and thelike. These latter types of compositions are not “unique” as that termis defined and used herein, i.e., they are “common.” In other words,they are calibrating or control genes whose function is not to tellwhether a particular “key” gene of interest is expressed, but rather toprovide other useful information, such as background or basal level ofexpression. The percentage of samples which are made of uniqueoligonucleotides or polynucleotide that correspond to the same type ofgene is generally at least about 30%, and usually at least about 60% andmore usually at least about 80%. Preferably, the arrays of the presentinvention will be of a specific type, where representative array typesinclude: human arrays, mouse arrays, cancer arrays, apoptosis arrays,human stress arrays, oncogene and tumor suppressor arrays, cell-cellinteraction arrays, cytokine and cytokine receptor arrays, rat arrays,blood arrays, mouse stress arrays, neuroarrays, and the like.

With respect to the oligonucleotide and/or polynucleotide compositionsthat correspond to a particular type or kind of gene, type or kind canrefer to a plurality of different characterizing features, where suchfeatures include: species specific genes, where specific species ofinterest include eukaryotic species, such as mice, rats, rabbits, pigs,primates, humans, etc.; function specific genes, where such genesinclude oncogenes, apoptosis genes, cytokines, receptors, proteinkinases, etc.; genes specific for or involved in a particular biologicalprocess, such as apoptosis, differentiation, cell cycle regulation,cancer, aging, proliferation, etc.; location specific genes, wherelocations include organs, such as heart, liver, prostate, lung etc.;tissue, such as nerve, muscle, connective, etc.; cellular, such asaxonal, lymphocytic, etc.; or subcellular locations, e.g., nucleus,endoplasmic reticulum, Golgi complex, endosome, lyosome, peroxisome,mitochondria, cytoplasm, cytoskeleton, plasma membrane, extracellularspace; specific genes that change expression level over time, e.g.,genes that are expressed at different levels during the progression of adisease condition, such as prostate genes which are induced or repressedduring the progression of prostate cancer.

In a preferred embodiment, longer oligonucleotides, preferably from80-300 nt in length, more preferably from 100-200 nt in length, are usedon the arrays. These are especially useful in place of cDNAs fordetermining the presence of mRNA in a sample, as the modifiedoligonucleotides have the advantage of rapid synthesis and purificationand analysis prior to attachments to the substrate surface. Inparticular, oligonucleotides with 2′ modified sugar groups showincreased binding affinity with RNA, and these oligonucleotides areparticularly advantageous in identifying mRNA in a sample exposed to anarray.

The length of the modified oligonucleotides allows the compositions tobind with the same affinity as a much longer unmodified nucleic acid,e.g. an unmodified cDNA. In the case where additional complementarity isneeded to certain domains or regions found in cDNA, multipleoligonucleotides may be used. Multiple oligonucleotides directed at aparticular gene or RNA molecule may be interspersed in a single region,or the different oligonucleotides may each be in a discrete region, e.g.to determine presence or absence of related molecules in a sample.

As mentioned above, the arrays of the present invention typicallycomprise one or more additional associated oligonucleotide compositionwhich does not correspond to the array type, i.e., the type or kind ofgene represented on the array. In other words, the array may compriseone or more compositions that are made of non “unique” oligonucleotides,e.g., oligonucleotides corresponding to commonly expressed genes. Forexample, compositions comprising oligonucleotides that bind to plasmidand bacteriophage oligonucleotides, oligonucleotides which bind to genesfrom the same or another species which are not expressed and do notcross-hybridize with the test nucleic acid, and the like, may be presentand serve as negative controls. In addition, compositions comprisinghousekeeping genes and other control genes from the same or anotherspecies may be present, e.g., to serve in the normalization of mRNAabundance and standardization of hybridization signal intensity in thesample assayed with the array.

Patents and patent applications describing arrays of oligonucleotidesand methods for their fabrication include: U.S. Pat. Nos. 5,242,974;5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327;5,445,934; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501;5,556,752; 5,561,071; 5,599,895; 5,624,711; 5,639,603; 5,658,734;5,700,637; 5,744,305; 5,837,832; 5,843,655; 5,861,242; 5,874,974;5,885,837; WO 93/17126; WO 95/11995; WO 95/35505; EP 742 287; and EP 799897. Patents and patent applications describing methods of using arraysin various applications include: U.S. Pat. No. 5,143,854; 5,288,644;5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270;5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,848,659; 5,874,219; WO95/21265; WO 96/31622; WO 97/10365; WO 97/27317; EP 373 203; and EP 785280. References that disclose the synthesis of arrays and reagents foruse with arrays include: Matteucci M. D. and Caruthers M. H., J. Am.Chem. Soc. (1981) 103:3185-3191; Beaucage S. L. and Caruthers M. H.,Tetrahedron Letters, (1981) 22(20):1859-1862; Adams S. P. et al., J. Am.Chem. Soc. (1983) 105:661-663; Sproat D. S. and Brown D. M., NucleicAcids Research, (1985) 13(8):2979-2987; Crea R. and Horn T., NucleicAcids Research, (1980) 8(10):2331-48; Andrus A. et al., TetrahedronLetters, (1988) 29(8):861-4; Applied Biosystems User Bulletin, Issue No.43, Oct. 1, 1987, “Methyl phosphonamidite reagents and the synthesis andpurification of methyl phosphonate analogs of DNA”; Miller P. S. et al.,Nucleic Acids Research, (1983) 11:6225-6242. Each of these isincorporated herein by reference as exemplary methods of constructionand use of arrays of the present invention. The methods of thesepublications can be readily modified to produce the arrays of theinvention with the modified oligonucleotides of the invention on theirsurface.

In a preferred embodiment, the modified oligonucleotides for use withthe present invention are synthesized prior to attachment onto thesubstrate. This affords the advantages that: (1) oligonucleotides ofknown composition and sequence can be produced; (2) oligonucleotides canbe analyzed and purified prior to attachment, which eliminates“shortmers,” i.e., oligonucleotides with insufficient length and/orincorrect sequence; (3) the methods used to produce oligonucleotides areless prone to error than current methods for production of cDNA, e.g.PCR with Taq polymerase, and (4) attachment to the substrate may bemonitored or assayed without destroying the array.

Numerous methods can be used for attachment of the oligonucleotides ofthe invention to the substrate. For example, modified oligonucleotidescan be attached using the techniques of, for example U.S. Pat. No.5,807,522, which is incorporated herein by reference for teachingmethods of polymer attachment. Other similar methods may be used, aswill be apparent to one skilled in the art upon reading the presenttechnology.

Use of Arrays of the Invention

Oligonucleotide and/or polynucleotide arrays provide a high throughputtechnique that can assay a large number of polynucleotides in a sample.A variety of different array formats have been developed and are knownto those of skill in the art. The arrays of the subject invention finduse in a variety of applications, including gene expression analysis,drug screening, mutation analysis and the like.

Arrays can be used, for example to examine differential expression ofgenes and can be used to determine gene function. For example, arrayscan be used to detect differential expression of a polynucleotidebetween a test cell and control cell (e.g., cancer cells and normalcells). For example, high expression of a particular message in a cancercell, which is not observed in a corresponding normal cell, can indicatea cancer specific gene product. Exemplary uses of arrays are furtherdescribed in, for example, Pappalarado et al., Sem. Radiation Oncol.8:217 (1998), and Ramsay, Nature Biotechnol. 16:40 (1998).

Methods for analyzing the data collected from hybridization to arraysare well known in the art. For example, where detection of hybridizationinvolves a fluorescent label, data analysis can include the steps ofdetermining fluorescent intensity as a function of substrate positionfrom the data collected, removing outliers, i.e., data deviating from apredetermined statistical distribution, and calculating the relativebinding affinity of the test nucleic acids from the remaining data. Theresulting data can be displayed as an image with the intensity in eachregion varying according to the binding affinity between associatedoligonucleotides and/or polynucleotides and the test nucleic acids.

Oligonucleotides having a sequence unique to that gene are preferablyused in the present invention. Different methods may be employed tochoose the specific region of the gene to be targeted. A rational designapproach may also be employed to choose the optimal oligonucleotidesequence for the hybridization array. Preferably, the region of the genethat is selected is chosen based on the following criteria. First, thesequence that is chosen should yield an oligonucleotide composition thatpreferably does not cross-hybridize with any other oligonucleotidecomposition present on the array. Second, the sequence should be chosensuch that the oligonucleotide composition has a low probability ofcross-hybridizing with an oligonucleotide having a nucleotide sequencefound in any other gene, whether or not the gene is to be represented onthe array from the same species of origin, e.g., for a human array, thesequence will not be present in any other human genes. As such,sequences that are avoided include those found in: highly expressed geneproducts, structural RNAs, repeated sequences found in the sample to betested with the array and sequences found in vectors. A furtherconsideration is to select oligonucleotides with sequences that providefor minimal or no secondary structure, structure which allows foroptimal hybridization but low non-specific binding, equal or similarthermal stabilities, and optimal hybridization characteristics.

Exemplary Array Types of the Invention

A variety of specific array types are also provided by the subjectinvention. As discussed above, array type refers to the nature of theoligonucleotide and/or polynucleotide compositions present on the arrayand the types of genes to which the associated compositions correspond.These array types include, but are not limited to: human array; mousearray; developmental array; cancer array; apoptosis array; oncogene andtumor suppressor array; cell cycle gene array; cytokine and cytokinereceptor array; growth factor and growth factor receptor array;neuroarrays; and the like.

In certain embodiments of the human array, human genes that may berepresented by the composition on the arrays include those for: (a)oncogenes and tumor suppressors; (b) cell cycle regulators; (c) stressresponse proteins; (d) ion channel and transport proteins; (e)intracellular signal transduction modulators and effectors; (f)apoptosis-related proteins; (g) DNA synthesis, repair and recombinationproteins; (h) transcription factors and general DNA binding proteins;(i) growth factor and chemokine receptors; (j) interleukin andinterferon receptors; (k) hormone receptors; (l) neurotransmitterreceptors; (m) cell surface antigens and cell adhesion proteins; (n)growth factors, cytokines and chemokines; (o) interleukins andinterferons; (p) hormones; (q) extracellular matrix proteins; (r)cytoskeleton and motility proteins; (s) RNA processing and turnoverproteins; (t) post-translational modification, trafficking and targetingproteins; (u) protein turnover; and (v) metabolic pathway proteins.

The arrays of the invention can be used in, among other applications,differential gene expression assays. Thus, arrays are useful in thedifferential expression analysis of: (a) diseased and normal tissue,e.g., neoplastic and normal tissue, (b) different tissue or tissuetypes; (c) developmental stage: (d) response to external or internalstimulus; (e) response to treatment; and the like. The arrays are alsouseful in broad scale expression screening for drug discovery andresearch, such as the effect of a particular active agent on theexpression pattern of genes in a particular cell, where such informationcan be used to reveal drug toxicity, carcinogenicity, etc.,environmental monitoring, disease research and the like.

Hybridization and Detection

Following preparation of the test nucleic acids from the tissue or cellof interest, the test sample is contacted with the array underhybridization conditions, where such conditions can be adjusted, asdesired, to provide for an optimum level of specificity in view of theparticular assay being performed. In analyzing the differences in thepopulation of labeled test binding agents generated from two or morephysiological sources using the arrays described above, each populationof labeled test samples are separately contacted to identical arrays ortogether to the same array under conditions of hybridization, preferablyunder stringent hybridization conditions (for example, at 50° C. orhigher and 0.1×SSC (15 mM sodium chloride/01.5 mM sodium citrate)), suchthat test nucleic acids hybridize to complementary oligonucleotidesand/or polynucleotides on the substrate surface.

Where all of the test nucleic acids have the same label, differentarrays can be employed 25 for each physiological source. Preferably, thesame array can be employed sequentially for each physiological source,with test samples removed from the array as described below.Alternatively, where the labels of the test nucleic acids are differentand distinguishable for each of the different physiological sourcesbeing assayed, the opportunity arises to use the same array at the sametime for each of the different test populations. Examples ofdistinguishable labels are well known in the art and include: two ormore different emission wavelength fluorescent dyes, like Cy3 and Cy5,two or more isotopes with different energies of emission, like ³²P and³³P, labels which generate signals under different treatment conditions,like temperature, pH, treatment by additional chemical agents, etc., orgenerate signals at different time points after treatment. Using one ormore enzymes for signal generation allows for the use of an even greatervariety of distinguishable labels, based on different substratespecificity of enzymes (e.g., alkaline phosphatase/peroxidase).

Following hybridization, non-hybridized labeled nucleic acid is removedfrom the support surface, conveniently by washing, generating a patternof hybridized oligonucleotide and/or polynucleotide on the substratesurface. A variety of wash solutions are known to those of skill in theart and may be used. The resultant hybridization patterns of labeled,hybridized oligonucleotides and/or polynucleotides may be visualized ordetected in a variety of ways, with the particular manner of detectionbeing chosen based on the particular label of the test nucleic acid,where representative detection means include scintillation counting,autoradiography, fluorescence measurement, colorimetric measurement,light emission measurement and the like.

Following detection or visualization, the hybridization patterns may becompared to identify differences between the patterns. Where arrays inwhich each of the different oligonucleotides and/or polynucleotidescorresponds to a known gene are employed, any discrepancies can berelated to a differential expression of a particular gene in thephysiological sources being compared.

Clearing of Test Nucleic Acids From Array

Following binding and visualization of a test sample on an array, thearray may be treated to remove the bound test nucleic acids. Theassociated nucleic acid compositions remain intact following treatment,allowing reuse of the treated array. The array of the inventionsubstantially retains its binding capabilities, and any differences inbinding ability may be determined using control sequences associated onthe array. Preferably, the array of the invention retains at least 75%of its binding capabilities, more preferably the array retains at least85% of its binding capabilities, and even more preferably the array ofthe invention retains at least 95% of its binding capabilities.

Arrays with associated modified oligonucleotide and/or polynucleotidecompositions can be exposed to a low pH environment, e.g., pH from0.5-4.5, which results in the degradation of non-modified nucleic acids.Following the treatment, the arrays of the invention are rinsed toremove any unwanted test nucleic acid fragments, residual label and thelike, and the arrays are prepared for reuse.

After detection of the test sample is complete, the array may beregenerated by removal and/or degradation of the test sample. Forexample, a two hour incubation of the sample-bound array in an acidsolution at pH 1.5, 39° C., results in complete loss of a full-lengthunmodified 14-mer oligonucleotide. Under these conditions the boundarray oligonucleotides of the invention maintain full length structuralintegrity. Following the acid incubation, a variety of wash conditionsmay be used to clear the test sample from the probe array. For example,increased temperature incubation of a low salt wash solution wouldresult in the dissociation of short test fragments from the array.Alternatively, a chemical denaturant (e.g., urea) could be used as awash to remove the test sample. Additional steps, such as an alkalinesolution rinse may also be added to the protocol to speed up the cycletime for regeneration.

The above-described washes and rinses can be avoided if the acidincubation is increased resulting in almost complete degradation of thetest sample under conditions where the array probe maintains itsintegrity. Actual incubation times required will vary somewhat fromarray type to array type, and may be shorter than those given below. Asa consequence of the degradation of the test sample the array probe/testsample hybrids become unstable under experimental conditions and may beremoved using rinses of the hybridization or stringent wash buffer.

Exemplary clearing conditions for use with the arrays of the inventionare:

-   -   (1) Incubation of the bound array with pH 1-2 acid solution, 8        hours at 39° C. Follow with three rinses at 39° C. with        stringent wash buffer, 0.1×SSC pH 7.0, and two rinses with        hybridization buffer, pH approximately 7.0. These two solutions        are for removal of degraded sample and the regeneration of the        substrate array and hence do not require a low pH. Array may        then be reused.    -   (2) Incubation of the bound array with pH 1-2 acid solution, 4        hours at 39° C. Follow with three 15 minute rinses at 39° C.        with 8.0 molar urea. Rinse once with stringent wash buffer, and        twice with hybridization buffer. Array can be reused at this        point.    -   (3) Incubation of the bound array with pH 1-2 acid solution, 4        hours at 39° C. Rinse twice at 39° C. with stringent wash        buffer. Incubate 20 minutes in 60° C. stringent wash buffer, and        rinse twice more with 60° C. stringent wash buffer. Rinse twice        with hybridization buffer. Array can be reused at this point.    -   (4) Incubation of the bound array with pH 1-2 acid solution, 4        hours at 39° C. Rinse twice with stringent wash buffer. Wash        twice with 39° C. alkaline solution for 15 minutes followed by        two washes with stringent wash buffer. Incubate 20 minutes in        60° C. stringent wash buffer. Rinse twice more with 60° C.        stringent wash buffer, and twice with hybridization buffer.        Array can be reused at this point.    -   (5) Incubation of the bound array with nuclease (actual        conditions vary with nuclease type) at 37° C. for 1 hour. Wash        twice with protein denaturing solution for 20 minutes. Rinse        twice with stringent wash buffer. Incubate 20 minutes in 60° C.        stringent wash buffer. Rinse twice with 60° C. stringent wash        buffer. Rinse twice with hybridization buffer. Array can be        reused at this point.    -   (6) Incubation of the bound array with pH 10-13 base solution        (e.g., NaOH) at room temperature for 1-30 minutes followed by        additional washes with pH 10-13 base solutions, water, or acidic        solution washes followed by a buffer wash.

Following treatment, the associated acid stable oligonucleotides of thearray remain 1) associated to the substrate surface; 2) structurallyintact; and 3) capable of binding with another test binding partner.

In addition, as an alternative way, arrays with associatedoligonucleotides characterized as nuclease resistant may be treated witha nuclease to remove bound test nucleic acids and label. The nucleaseused can be chosen depending on the nature of the binding between theassociated oligonucleotide and/or polynucleotide and the molecules ofthe test sample and the attachment of the oligonucleotide and/orpolynucleotide to the array. For example, if the associatedoligonucleotides are end-blocked oligonucleotides, and the test sampleis comprised of mRNA molecules, then the appropriate nuclease would beone that recognizes RNA-DNA hybrids, e.g., Ribonuclease H. In anotherexample, if the associated oligonucleotides are end-blockedoligonucleotides, and the test sample is comprised of cDNA molecules,then the appropriate nuclease would be one that recognizes doublestranded DNA complexes, e.g., Deoxyribonuclease I or II, andExodeoxyribonuclease III or V. In yet another example, if the associatedoligonucleotides are end-blocked cRNA and the test sample is comprisedof mRNA, the appropriate nuclease is one that recognizes RNA-RNAhybrids, such as micrococcal nuclease. Similarly, nucleases that are 5′or 3′ specific may be chosen depending on the attachment site of theoligonucleotide and/or polynucleotide to the array. Since theoligonucleotides of this embodiment of the invention arenuclease-resistant, the test samples will be specifically targeted anddegraded by the nuclease.

Actual choice of regeneration conditions should take into considerationthe type of substrate, the type of attachment of probe to substrate,test sample type, and whether there are clearing time constraints. Incases where the substrate is acid sensitive it would be moreadvantageous to use nuclease digestion to remove the test sample fromthe array. Such modifications would be well within the skill of one inthe art upon reading the present disclosure and description of thesubject arrays.

Kits Having Arrays of Present Invention

Also covered are kits for performing analyte binding assays using thearrays of the present invention. Such kits according to the subjectinvention will at least comprise the arrays of the invention havingassociated modified oligonucleotides and/or polynucleotides. Kits alsopreferably comprise an agent for removal of test binding agents, e.g., asolution with low pH and/or with nuclease activity. The kits may furthercomprise one or more additional reagents employed in the Variousmethods, such as: 1) prime for generating test nucleic acids; 2) dNTPsand/or rNTPs (either premixed or separate), optionally with one or moreuniquely labeled dNTPs and/or rNTPs (e.g., biotinylated or Cy3 or Cy5tagged dNTPs); 3) post synthesis labeling reagents, such as chemicallyactive derivatives of fluorescent dyes; 4) enzymes, such as reversetranscriptases, DNA polymerases, and the like; 5) various buffermediums, e.g., hybridization and washing buffers; 6) labeled probepurification reagents and components, like spin columns, etc.; and 7)signal generation and detection reagents, e.g., streptavidin-alkalinephosphatase conjugate, chemifluorescent or chemiluminescent substrate,and the like.

EXAMPLES

The present invention and its particular embodiments are illustrated inthe following examples. The examples are not intended to limit the scopeof this invention but are presented to illustrate and support the claimsof this present invention.

Example 1 Synthesis and Purification of Modified Nucleic Acids

Oligonucleotides were synthesized using commercial phosphoramidites oncommercially purchased DNA synthesizers from <1 uM to >1 mM scales usingstandard phosphoramidite chemistry and methods that are well known inthe art, such as, for example, those disclosed in Stec et al., J. Am.Chem. Soc. 106:6077-6089 (1984), Stec et al., J. Org. Chem.50(20):3908-3913 (1985), Stec et al., J. Chromatog. 326:263-280 (1985),LaPlanche et al., Nuc. Acid. Res. 14(22):9081-9093 (1986), and Fasman,Practical Handbook of Biochemistry and Molecular Biology, 1989, CRCPress, Boca Raton, Fla., herein incorporated by reference.

Oligonucleotides were deprotected following phosphoramiditemanufacturer's protocols. Unpurified oligonucleotides were either drieddown under vacuum or precipitated and then dried. Sodium salts ofoligonucleotides were prepared using the commercially available DNA-Mate(Barkosigan Inc.) reagents or conventional techniques such ascommercially available exchange resin, e.g., Dowex, or by addition ofsodium salts followed by precipitation, diafiltration, or gelfiltration, etc.

A variety of standard methods were used to purify and produce thepresently described oligonucleotides. In brief, oligonucleotides werepurified by chromatography on commercially available reverse phase (forexample, see the RAININ Instrument Co., Inc. instruction manual for theDYNAMAX}-300A, Pure-DNA reverse-phase columns, 1989, or current updatesthereof, herein incorporated by reference) or ion exchange media such asWaters' Protein Pak or Pharmacia's Source Q (see generally Warren andVella, 1994, “Analysis and Purification of Synthetic Nucleic Acids byHigh-Performance Liquid Chromatography”, in Methods in MolecularBiology, vol. 26; Protocols for Nucleic Acid Conjugates, S. Agrawal, Ed.Humana Press, Inc., Totowa, N.J.; Aharon et al., 1993, J. Chrom.698:293-301; and Millipore Technical Bulletin, 1992, Antisense DNA:Synthesis, Purification, and Analysis). Peak fractions were combined andthe samples were concentrated and desalted via alcohol (ethanol,butanol, isopropanol, and isomers and mixtures thereof, etc.)precipitation, reverse phase chromatography, diafiltration, or gelfiltration or size-exclusion chromatography.

Lyophilized or dried-down preparations of oligonucleotides weredissolved in pyrogen-free, sterile, physiological saline (i.e., 0.85%saline), sterile Sigrna water, and filtered through a 0.45 micron Gelmanfilter.

Example 2 Stability of Modified Oligonucleotide Duplexes

The stability of duplexes having 2′-substituted nucleotides versusduplexes without such modification was tested by examining the T_(m) ofthese complexes. 4 μM each of 20-mer oligonucleotide (5′ -ggt ggt tcctcc tca gtc gg-3′; SEQ ID NO:1) and its complement (5′-ccg act gag aaggaa cca cc-3′) were bound in a solution of 50 mM NaCl, 10 mM PO4 buffer,pH 7.4. Each of the nucleotides of the oligonucleotide had the same 2′group. Following binding, the melting temperature was determined asdescribed. (See L. L. Cummins et al., Nucleic Acids Research23:2019-2024 (1995).

Results were as follows: SEQ ID NO: 1 SEQ ID NO: 2 T_(m) Regular RNA andRegular DNA 66° C. Regular RNA and 2′-O-methyl 79° C. Regular DNA andp-ethoxy DNA 55° C. Regular RNA and p-ethoxy RNA 56° C. Regular RNA andp-ethoxy 2′-O-methyl 71° C.

The duplexes with the 2′-O-methyl substitutions display a significantlyincreased T_(m) compared to RNA or DNA with a 2′ H or 2′ OH,respectively. RNA or DNA with propyl or fluoro substitutions at the 2′position display an even higher T_(m) than does the 2′-O-methyl.

Example 3 Acid Stability of the Oligonucleotides of the Invention

Homopolymers of 2′-O-methyl A, C, G, and U twelve bases long, weresynthesized with 3′ and 5′ inverted T-blocked ends. They were purified,desalted, lyophilized, and dissolved at 300 A₂₆₀ per ml in sterilewater. Samples were removed and diluted 1 to 4 with either 0.1 N HCl or1.0 N HCl to give final pHs of approximately 1 and 0, respectively, andplaced in a heat block at 39° C. Aliquots were taken at 0, 2, 4 and 24hours, diluted 1:20 into a solution of 0.025 M NaOH and 0.03 M NaCl,stored at −20° C. until being run on an analytical HPLC under stronglydenaturing conditions on an anion exchange column. % Full LengthHomopolymer pH 0 hr 2 hr 4 hr 24 hr A 1 99 99 99 99 C 1 99 99 99 96 G 196 98 98 98 U 1 97 — 97 97 A 0 99 99 99 99 C 0 99 99 98 97 G 0 96 97 9789 U 0 97 — 97 96

It was evident that there is essentially no degradation at pH 1 and 39°C. and only slight degradation over 24 hours at pH 0 and 39° C.

Example 4 Acid Stability of the Oligonucleotides of the Invention

A 14 mer heteropolymer was synthesized as a regular phosphodiester DNA(O), a phosphorothioate DNA (S), an unblocked 2′-O-methyl RNA (2′ om), a2′-O-methyl RNA with 3′ and 5′ butanol blocked ends (B2′ om), and aphosphorothioate chimera having four 2′-O-methyl phosphorothioate baseson either side of 6 interior phosphorothioate DNA bases (SD). They werepurified, desalted, lyophilized, and dissolved at 300 A₂₆₀ per ml insterile water. Samples were removed and diluted 1 to 4 with 0.1 N HCl togive a final pH of approximately 1.5, and placed in a heat block at 39°C. Aliquots were taken at the times indicated and diluted 1:20 into asolution of 0.025 M NaOH and 0.03 M NaCl, and were run on an analyticalHPLC under strongly denaturing conditions on an anion exchange column.Initially all but the end-blocked 2′-O-methyl RNA solutions becamecloudy upon addition of the HCl. Upon heating, both the phosphodiesterDNA and the unblocked 2′-O-methyl RNA became clear. The twooligonucleotides with phosphorothioate linkages appeared cloudy untilabout 2 hours when they slowly began to clear as they decomposed. % FullLength Oligo 0 hr 0.5 hr 1.0 hr 2 hr 4 hr 6 hr 1 d 2 d 3 d 5 d 10 d 20 dO 99 38 10 0 0 0 0 — — — — — S 95 65 29 1 0 0 0 — — — — SD 97 83 70 49 00 0 — — — — — 2′om 99 99 99 99 98 98 98 96 94 94 87 80 B2′om 100 100 100100 99 99 98 97 97 95 90 81

The 2′-O-methyl oligonucleotides, both unblocked and blocked, are farmore stable than the corresponding phosphodiester, phosphorothioate, ora mixed 2′-O-methyl phosphorothioate structure that Agrawal et al.recommended to increase bioavailability.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. An array comprising a plurality of modified oligonucleotidecompositions stably associated with the surface of a support, whereineach oligonucleotide composition is characterized by: an oligonucleotidebackbone structure modified from that of a naturally occurringnucleotide polymer; wherein the oligonucleotides of the composition arecharacterized by a binding affinity greater than that of acorresponding, non-modified oligonucleotide.
 2. The array of claim 1,wherein the oligonucleotides are comprised of a modification at the 2′site of the sugar group of at least one nucleotide.
 3. The array ofclaim 1, wherein the oligonucleotides are comprised of at least onemodified internucleoside linkage.
 4. The array of claim 1, wherein saidmodified oligonucleotides have an average length of from about 80 toabout 300 nucleotides.
 5. The array of claim 1, wherein said modifiedoligonucleotides have an average length of from about 100 to about 200nucleotides.
 6. The array of claim 1, wherein oligonucleotides of eachof said oligonucleotide compositions has a different sequence fromoligonucleotides of any other oligonucleotide composition on the array.7. The array of claim 1, wherein each oligonucleotide compositioncomprises a population of identical oligonucleotides.
 8. The array ofclaim 1, wherein each oligonucleotide composition comprises a pluralityof oligonucleotides that bind to a particular nucleic acid.
 9. The arrayof claim 1, wherein the number of oligonucleotide compositions on saidarray ranges from about 2 to about 10⁹.
 10. An array comprising aplurality of modified oligonucleotide compositions stably associatedwith the surface of a support, wherein each oligonucleotide compositionis characterized by: an oligonucleotide backbone structure modified fromthat of a naturally occurring nucleotide polymer; wherein theoligonucleotides of the composition is characterized by a pH stabilityof at least one hour at 37° C. at a pH in a range of about 0.5 to about10.
 11. The array of claim 10, further comprising a blocking chemicalmodification at or near at least one end of said oligonucleotide,wherein the oligonucleotide is further characterized by having anuclease resistance of at least twice that of a naturally occurringoligonucleotide having the same sequence and number of bases.
 12. Thearray of claim 10, wherein the oligonucleotide is stable at a pH of from0.5 to 6.0.
 13. The array of claim 10, wherein the modifiedoligonucleotide is further characterized by modification of at least 25%of the intemucleoside linkages of the oligonucleotide.
 14. An arraycomprising a plurality of oligonucleotide compositions stably associatedwith the surface of a support, wherein each oligonucleotide compositionis characterized by: an oligonucleotide backbone structure modified fromthat of a naturally occurring nucleotide polymer; and a blockingchemical modification at or near at least one end of theoligonucleotide; wherein the oligonucleotide is characterized by anuclease resistance of at least twice that of a naturally occurringpolymer having the same number of nucleotides.
 15. An array of modifiedoligonucleotides, the array comprising: a planar, non-porous solidsupport having a surface; a plurality of different modifiedoligonucleotides attached to the surface of the solid support at adensity exceeding 400 different modified oligonucleotides/cm², whereineach of the different modified oligonucleotides is attached to thesurface of the solid support in a different predefined region, has adifferent determinable sequence, and is at least 80 nucleotides inlength; and further wherein the modified oligonucleotides arecharacterized by a characteristic selected from the group consisting of(a) a binding affinity of at least about 1.25 times that of acorresponding, non-modified oligonucleotide, (b) a pH stability of atleast one hour at 37° C. at a pH in a range of about 0.5 to 10; and (c)a nuclease resistance of at least twice that of a naturally occurringoligonucleotide having the same sequence and number of bases. 16-21.(canceled)